Prohibitin as Target for Cancer Therapy

ABSTRACT

The present invention relates to pharmaceutical compositions comprising inhibitors of Prohibitin (PHB) for the prevention or/and treatment of hyperproliferative disorders.

The present invention relates to pharmaceutical compositions comprising inhibitors of Prohibitin (PHB) for the prevention or/and treatment of hyperproliferative disorders. Further, the present invention concerns PHB inhibitors and screening methods for identification of PHB inhibitors, which inhibitors are suitable for prevention or/and treatment of hyperproliferative disorders.

Ras-MAPK cascade has been activated in almost all tumours and plays a very important role in proliferation, tumour cell migration, invasion of the extracellular matrix, resistance to apoptosis and angiogenesis (ability to induce new blood vessel formation). There are at least 20 new therapeutic agents in clinical trials at present exploiting the interaction and activation of several components of this highly conserved pathway. This includes a range of Farnesyl transferase inhibitors (FTIs) to block the translocation of Ras to the plasma membrane, for example the drug R115777 (Zanestra is in clinical trail phase III), SCH66336 (SARASAR), to antisense oligonucleotides to Raf and H-Ras (ISIS5132 and ISIS25403) from Isis pharmaceuticals. Apart from targeting the Ras-MAPK cascade there are also several drugs targeting upstream pathways most importantly the EGF receptor family members. As EGFR or its family members is found to be highly expressed in more than 50% of carcinomas. Most importantly Her-2, an EGFR family member is amplified and therefore the causative for breast cancer. The drugs which target these members range from kinase inhibitors like the OSI-774 (Tarceva) to the well known Herceptin (Transtuzumab, a humanized antibody ) which is now licensed for use on breast cancer. The major aim of these drugs is to reduce the hyper activation of Ras-MAPk kinase cascade and sensitize them to chemotherapeutic cancer drugs. As we have identified PHB as a new modulator of this known pathway, targeting PHB could be a potential alternative in all these cases.

Ras proteins control signalling pathways responsible for normal growth and malignant transformation. Raf protein kinases are direct Ras effector proteins initiating the mitogen-activated protein (MAP) kinase cascade leading to the activation of transcription factors via ERK. Here we show, that prohibitin (PHB), a ubiquitously expressed and evolutionarily conserved protein is indispensable for the activation of the Raf-MAP kinase pathway by Ras. Raf kinase fails to interact with active Ras induced by epidermal growth factor (EGF) in the absence of prohibitin. Prohibitin and Raf kinase are enriched in caveolae and after depletion of prohibitin, Raf kinase is lost from this compartment. Constitutively active Raf kinase induces ERK activation independent of prohibitin. Interestingly, we find a prominent role of the prohibitin dependent branch of the Ras signalling pathway in epithelial cell adhesion and migration. In prohibitin deficient cells the adherent complex proteins cadherin and β-catenin relocalise to plasma membrane and thereby stabilize adherent junctions. Our data show an unexpected role of prohibitin in the activation of the Ras-Raf signalling pathway and in modulating epithelial cell adhesion and migration.

Prohibitin (PHB) is involved in diverse cellular processes like proliferation and energy metabolism and is found in different cellular compartments including the nucleus and mitochondria (1). We identified PHB in a RNA interference based loss of function screen for proteins involved in the regulation of apoptosis (Machuy et al., in press). Besides the slight sensitisation for apoptotic stimuli observed (not shown), the most prominent phenotype of HeLa cells transfected with siRNAs to suppress PHB expression (siPHB) was reduced spreading and increased intercellular adhesion to form tiny islands of densely packed cells in place of a uniform monolayer (FIG. 1). The observed phenotype correlated with prohibitin expression because, due to the transient effect of siRNAs, PHB expression was normal 6 days post transfection and the growth of epithelial cell as monolayer was restored (data not shown). In order to exclude unspecific effects of the siRNA used, an additional siRNA designed to target the coding region (siPHB-2) and one to target the 3′ untranslated region (siPHB-3) of the prohibitin mRNA were used. All siRNA, siPHB (FIG. 1A, B), siPHB-2 (FIG. S1) and siPHB-3 (FIG. 1E, F) efficiently interfered with the expression of prohibitin and induced a similar phenotype as siPHB (FIG. S1C, FIG. 1C, D). PHB expression was then complemented in siPHB-3 transfected cells by co-transfecting an expression plasmid harbouring the cloned PHB gene. Complemented cells formed monolayers suggesting that the formation of small islands depended on the lack of PHB (FIG. 1D). An similar phenotype could also be detected in other cell types like human larynx carcinoma (HEp-2) and gastric cancer cells (AGS) (not shown).

To investigate the nature of intercellular adhesion we performed scanning and transmission electron microscopy studies on cells with silenced PHB expression. FIG. 2(A-D) shows the SEM and TEM images of the control transfected and siPHB transfected cells. As observed previously, cells transfected with siPHB have almost no intercellular spaces and packed compactly when compared to the control transfected cells. To check if the increased intercellular adhesion was due to the formation and/or stabilization of adherens junctions (AJs), we performed confocal microscopy analysis in the PHB knock down cells. The formation and/or the stabilization of the AJs can be studied by monitoring the intracellular localizations of AJ complex proteins like cadherins or β-catenin. In contrast to control cells, PHB knock down cells exhibited a strong staining for both cadherins and β-catenin at the plasma membrane indicating a predominant lateral localization of these junctional proteins resulting in stabilized adherens junctions (FIG. 2) (E-J).

The control of adherens junction formation is an important step of malignant transformation in epithelial cells and has previously been shown to be controlled by receptor tyrosine kinases like the epithelial growth factor receptor (EGFR) and the human EGFR-related 2 (Her-2). Both receptors are deregulated in several epithelial tumours leading to constitutive kinase activity (2) und malignant transformation (3). Stimulation of these receptors with epidermal growth factor (EGF) results in the enhanced migration on collagen (4) or extracellular matrix a widely used in vitro model for studying cancer metastasis (5). HeLa cells lacking PHB expression were defective in such a migration assay (FIG. 2K) suggesting that PHB is required for EGF-induced migration.

We tested then whether down regulation of the Her-2 or EGFR resulted in the formation of cell clusters as previously observed for cells lacking PHB expression. Cells depleted of Her-2 (FIG. S2) or EGFR (not shown) formed cell clusters similar to those observed in cells with reduced PHB expression. Transfection of siPHB had no effect on the amount of surface exposed EGFR (not shown) or Her-2 (FIG. S2), suggesting that PHB is involved in a signalling pathway downstream of EGFR and Her-2.

EGFR and Her-2 signal via Ras proteins to different pathways involved in cell proliferation, migration, differentiation including the Raf-MAPK-ERK or the phosphoinositide 3-kinase (PI3K)-AKT pathway (2,6). Interestingly, depletion of Her-2 or PHB strongly reduced the phosphorylation of ERK but not of AKT suggesting a specific involvement of PHB in the Ras-Raf-MAPK pathway (FIG. 3A). Neither the protein levels of Ras, Raf-1, ERK2 (FIGS. 3 and 4) or MEK-1 (not shown) were reduced in PHB depleted cells ruling out an unspecific inhibitory effect of the transfected siRNA. Furthermore, expression of the cloned PHB gene in siPHB transfected cells restored the EGF-dependent phosphorylation of ERK showing a specific function of PHB in signalling from RTK receptors to ERK (FIG. 3H).

Raf-1 kinase is regulated by phosphorylation at several amino acids. Ser259 is the major target for inhibitory phosphorylation and dephosphorylation of Ser259 generally precedes the activating phosphorylation at the Ser338 (7). As expected, stimulation of HeLa cells with EGF caused an increased phosphorylation of Raf-1 at Ser338 (FIG. 3D). Interestingly, cells lacking PHB lacked the basal as well as the EGF induced phosphorylation at Ser338 indicative of a block in Raf-1 kinase activation by PHB depletion (FIG. 3D). Moreover, in the absence of PHB, no alteration or relatively high levels of Raf-1 phosphorylated at Ser259 were detected (FIG. 3D). Restoration of PHB expression by complementing the siPHB transfected cells with the cloned PHB gene restored the basal as well as EGF-dependent activation of Raf-1, apparent by high Raf-1 pSer338 and low Raf-1 pSer259 levels (FIG. 3D). Thus, PHB is required for EGF-induced Raf-1 activation.

If PHB is involved in the activation of Raf-1-MEK-ERK, cell clusters induced by reduced motility and stabilized adherens junctions should be affected by direct activation of Raf-1 or the expression of a constitutively active Raf-1 derivative. Treatment of siPHB transfected cells with phorbol myristate acetate (PMA), an activator of Raf-1 (8), rapidly resolved the cell clusters formed upon PHB depletion in between 2 to 4 hours upon addition (FIG. 3E; see also supplementary movie). As expected, PMA treated cells showed increased ERK phosphorylation irrespective of the presence or absence of PHB (FIG. 3F). Likewise, expression of a constitutive active Raf-1 mutant RafBXB (9) was sufficient to prevent clusters formation in cells with silenced PHB expression (FIG. 3G, H). RafBXB expression stimulated the phosphorylation of ERK in siPHB-3 transfected cells to a similar extent as the transgenic expression of the cloned PHB gene (FIG. 3G, H). Taken together, these data demonstrate a direct role of PHB in the activation of Raf-1.

Activation of Raf-1 requires the Ras-dependent recruitment to the plasma membrane (10,11) where both proteins reside in special caveolin rich patches called caveolae (12). PHB has previously been shown to localise to the plasma membrane and further a direct interaction of PHB and Raf-1 was also reported in U937 cells (13). We have also detected Raf-1, as well as Raf-1 pS338 in the PHB immunoprecipitates from Hela cells treated with or without EGF (FIG. S3). Interestingly, we also found PHB in caveolae together with Raf-1 (FIG. 4A). However, Raf-1 was not detected in the caveolae of siPHB treated cells (FIG. 4A), suggesting that PHB is required at a step prior to membrane recruitment of Raf-1.

The GTP-bound active form of Ras proteins directly binds and activates Raf-1 (14). We therefore asked whether Ras and Raf still interact in PHB depleted cells. As expected, endogenous Raf-1 interacted with Ras in EGF treated cells transfected with control siRNAs but not in untreated cells (FIG. 4B) demonstrating the activation dependent binding of Ras to Raf-1. Interestingly, the binding of Ras and Raf-1 was completely abrogated in cells lacking PHB (FIG. 4B) suggesting a direct role of PHB in the interaction of Ras and Raf-1. To confirm that PHB interferes with activation of Raf-1 by Ras we transfected constitutively active HA-Ras(G12V) in HeLa cells and either co-transfected with siPHB or control siRNAs. Interestingly, overexpressed active Ras(G12V) induced the activation of Raf-1 and ERK in control transfected cells but not in cells depleted of PHB (FIG. 4D). These results clearly show that PHB is required for the interaction of Raf-1 and Ras, a prerequisite for membrane translocation and activation of Raf-1.

We have demonstrated an unexpected role of PHB in the activation of Raf-1 by Ras. Moreover, the branch of the Ras signalling cascade controlled by PHB plays an important role in the motility of the cell. In fact, tumour cells with reduced PHB expression showed a dramatic redistribution of cadherin and β-catenin to the plasma membranes indicative of a conversion of the tumour cells from a transformed to a non-transformed phenotype (15). Moreover, prohibitin has been shown to be over expressed In gastric carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, and bladder carcinoma (16-20) indicating that prohibin may play a prominent role in the progression of neoplastic carcinoma. The facts that activating Ras mutations are found in more than 20% of all tumours (21) and that the highly conserved PHB is essential for signalling via Ras imposes PHB as a possible target for tumour therapy. One strategy could depend on the specific interference with the binding of PHB to Raf kinases in order to block activation by oncogenic Ras proteins and cellular transformation.

In one embodiment of the present invention, cancers with high expression of EGFR or Ras point mutations (pancreas, lung adenocarcinoma, colorectal, thyroid, bladder, liver and kidney) can be treated or prevented by decreasing the PHB activity in the method of the present invention for treating or/and prevention of hyperprolerative disorders. In some of these cancers, PHB is overexpressed. Since increased PHB levels are found in metastases, metastatic tumours can also be treated by decreasing PHB activity.

Subject of the present invention is also treatment of EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, or/and Raf-1 transformed tumours. Since PHB signals downstream of EGFR and Her-2, also EGFR overexpressing tumours and Her-2 family overexpressing tumours can be treated by inhibition of PHB or/and downregulation of PHB.

Since Herceptin acts on Her-2, PHB inhibition may be an alternative treatment strategy in Herceptin resistant tumours, acting e.g. via EGFR signalling which is a path alternative to the Her-2 path.

The involvement of PHB in the signalling cascade of Raf-1 or/and B-Raf leads to the conclusion that also Raf-1 (C-Raf) transformed tumours or/and B-Raf transformed tumours can be treated by inhibition or/and downregulation of PHB.

As we have identified that reducing the activity of PHB in tumour cells block the Raf-Mapk kinase cascade directly and efficiently, RNA interference, antisense nucleic acids or/and a chemical based approach can be employed to reduce PHB expression in tumour cells in the method of the present invention for treating or/and prevention of hyperprolerative disorders.

PHB inhibition in the context of the present invention includes downregulation of PHB transcription or/and translation. While not wishing to be bound to theory, the mechanism of PHB action may be targeting of PHB to membranes combined with targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes. Further, posttranslational modification may be important for the proper action of PHB. Therefore, PHB inhibition of the present invention also includes inhibition of targeting of PHB to membranes, inhibition of targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes, and inhibition of posttranslational modification of PHB, in particular of posttranslational modifications required for PHB targeting to membranes or/and required for Raf kinase, in particular Raf-1 targeting to membranes.

The PHB-Raf interaction is mediated by a sequence of about 20-30 amino acids, indicating that the interaction between PHB and Raf can be interrupted with either a small molecule inhibitor or a small peptide.

In one embodiment, the inhibitor of PHB of the present invention is a nucleic acid, which can be

-   -   (i) an RNA molecule capable of RNA interference,     -   (ii) a precursor of the RNA molecule (i), or     -   (iii) a DNA molecule encoding the RNA molecule (i) or the         precursor (ii).

RNA molecules capable of RNA interference are described in WO 02/44321 which is included by reference herein.

Preferably, the inhibitor of the present invention, in particular the nucleic acid of the present invention, is used in a pharmaceutical composition.

The antibody of the present invention may be a monoclonal or polyclonal antibody, a chimeric antibody, a chimeric single chain antibody, a Fab fragment or a fragment produced by a Fab expression library.

Techniques of preparing antibodies of the present Invention specific for PHB are known by a skilled person. Monoclonal antibodies may be prepared by the human B-cell hybridoma technique or by the EBV-hybridoma technique (Köhler et al., 1975, Nature 256:495-497, Kozbor et al., 1985, J. Immunol. Methods 81,31-42, Cote et al., PNAS, 80:2026-2030, Cole et al., 1984, Mol. Cell Biol. 62:109-120). Chimeric antibodies (mouse/human) may be prepared by carrying out the methods of Morrison et al. (1984, PNAS, 81:6851-6855), Neuberger et al. (1984, 312:604-608) and Takeda et al. (1985, Nature 314:452-454). Single chain antibodies may be prepared by techniques known by a person skilled in the art.

Recombinant immunoglobulin libraries (Orlandi et al, 1989, PNAS 86:3833-3837, Winter et al., 1991, Nature 349:293-299) may be screened to obtain an antibody of the present invention which are specific against PHB. A random combinatory immunoglobulin library (Burton, 1991, PNAS, 88:11120-11123) may be used to generate an antibody with a related specifity having a different idiotypic composition.

Another strategy for antibody production is the in vivo stimulation of the lymphocyte population.

Furthermore, antibody fragments (containing F(ab′)₂ fragments) of the present invention can be prepared by protease digestion of an antibody, e.g. by pepsin. Reducing the disulfide bonding of such F(ab′)₂fragments results in the Fab fragments. In another approach, the Fab fragment may be directly obtained from an Fab expression library (Huse et al., 1989, Science 254:1275-1281).

Polyclonal antibodies of the present invention may be prepared employing PHB or immunogenic fragments thereof as antigen by standard immunization protocols of a host, e.g. a horse, a goat, a rabbit, a human, etc., which standard immunization protocols are known by a person skilled in the art.

Fragments of polypeptides or/and peptides, in particular immunogenic fragments of SEQ.ID.NO:2 or a Raf kinase may have a length of at least 5 amino acid residues, preferably at least 10, more preferably at least 20 amino acid residues. The length of said fragments may be 200 amino acid residues at the maximum, preferably 100 amino acid residues at the maximum, more preferably 60 amino acid residues at the maximum, most preferably 40 amino acid residues at the maximum.

It can easily be determined by a skilled person if a gene is upregulated or downregulated. In the context of the present invention, upregulation of gene expression may be an upregulation by a factor of at least 2, preferably at least 4. Downregulation in the context of the present invention may be a reduction of gene expression by a factor of at least 2, preferably at least 4. Most preferred is essentially complete inhibition of gene expression.

“Reduction (increase) of the amount” may be a downregulation (upregulation) of gene expression by a factor of at least 2, preferably at least 4. In the case of reduction, essentially complete inhibition of gene expression is most preferred. Examples are reduction of the amount of PHB or reduction (increase) of the gene product amount of the genes of Table 1 or/and 2.

“Reduction (increase) of the activity” may be a decrease (increase) of activity of a gene or gene product by a factor of at least 2, preferably at least 4. In the case of activity reduction, essentially complete inhibition of activity is most preferred. An example relevant for the present invention is reduction of PHB activity or reduction (increase) of activity of the genes of Table 1 or/and 2.

A “target” or “target gene” for treatment of hyperproliferative disorders in the context of the present invention is a gene the expression of which is influenced by prohibitin inhibition. Prohibition inhibition may be provided by downregulation of PHB expression or by inhibition of PHB activity by an inhibitor of the present invention as discussed above.

The surprising finding of the present invention that PHB inhibition is a promising approach for treatment of hyperproliferative disorders leads to the conclusion that also genes which act downstream of PHB in the signalling cascade may be suitable targets for treatment of hyperproliferative disorders. Therefore, subject of the present invention is a method for identification of target genes for treatment of hyperproliferative disorders, based upon inhibition of PHB.

The term “target” also includes a gene product (RNA, in particular mRNA, tRNA, rRNA, a polypeptide or/and a protein) of the target gene. Preferred gene products of a target gene are selected from mRNA and a polypeptide or a protein encoded by the target gene. The most preferred gene product is a polypetide or protein encoded by the target gene. A protein or polypeptide of the present invention may be posttranslationally modified or not.

In the context of the present invention, “activity” of the gene or/and gene product includes transcription, translation, posttranslational modification, modulation of the activity of the gene product by ligand binding, which ligand may be an activator or inhibitor, etc.

In the case of PHB, “activity” also includes targeting of PHB to membranes, targeting of a Raf kinase, in particular of Raf-1 to membranes, posttranslational modification of PHB, in particular required for PHB or/and Raf kinase, in particular Raf-1 targeting to membranes.

Examples of genes which are upregulated by PHB inhibition are described in Table 1. Examples of genes which are downregulated by PHB inhibition are described in Table 2. The targets of Table 1 or/and 2 may be used for identification of new compounds for treatment or/and prophylaxis of hyperproliferative disorders. Therefore, yet another subject of the present invention is a screening method for identification of a compound suitable for treatment of a hyperproliferative disorder based upon the genes of Table 1 and 2.

The compound to be identified by the method of the present invention is a compound which increases the amount or/and the activity of the gene product of the at least one gene of Table 1 or decreases the amount or/and activity of the gene product of the at least one gene of Table 2. Therefore, the compound to be identified may be an inhibitor or an activator of a target gene or a gene product thereof.

The inhibitor or activator of a target gene or a gene product thereof may be selected from the group of nucleic acids, nucleic acid analogues such as ribozymes, peptides, polypeptides, and antibodies. A nucleic acid inhibitor or activator of a target gene or gene product thereof can be

-   -   (i) an RNA molecule capable of RNA interference,     -   (ii) a precursor of the RNA molecule (i), or     -   (iii) a DNA molecule encoding the RNA molecule (i) or the         precursor (ii).

The antibody may be an antibody specific for a gene product of a target gene, in particular an antibody specific for a polypeptide or protein encoded by a target gene. Production of a suitable antibody is described above in the context of prohibitin.

The inhibitor or activator of a target gene or gene product thereof may be used for the manufacture of a pharmaceutical composition for treatment or/and prophylaxis of a hyperproliferative disorder.

In the method of the present invention for identification of a compound suitable for treatment of a hyperproliferative disorder based upon the genes of Table 1 and 2, preferably employed are genes listed in Table 1 and 2 involved in cancer signalling, angiogenesis, adhesion, invasion or/and metastasis formation.

Most preferred genes listed in Table 1 and 2 are genes involved in cancer signalling: NM_(—)004419, DUSP5 dual specificity phosphatase 5, direct target of p53; U21049, DD96, epithelial protein up-regulated in carcinoma, membrane associated protein 17; NM_(—)005130 HBP17 (heparin-binding growth factor binding protein), which binds to HB-EGF which activates the RAS-signaling pathway.

Further most preferred genes listed in Table 1 and 2 are genes encoding angiogenis factors: NM_(—)003370, VASP, vasodilator-stimulated phosphoprotein, direct angiogenic activity, which plays also a role in cell motility and metastasis formation; NM_(—)139314, ANGPTL4, Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1; NM_(—)006108, SPON1, Homo sapiens spondin 1, extracellular matrix protein (SPON1), having a potential function as an angiopoietin or/and as spondin.

Further most preferred genes listed in Table 1 and 2 are genes involved in adhesion, invasion and metastasis: NM_(—)003255, TIMP2, tissue inhibitor of metalloproteinase 2, which plays an important role in invasion and metastasis formation by inhibiting metalloproteases; NM_(—)006108, SPON1, Homo sapiens spondin 1, extracellular matrix protein (SPON1); NM_(—)000213, ITGB4, integrin beta 4, Extracellular matrix binding and signaling, plays an important role in adhesion and invasion; NM_(—)012385 P8, p8 protein (candidate of metastasis 1); NM_(—)004360, CDH1, cadherin 1, type 1, E-cadherin (epithelial), plays an important role in adhesion and invasion as a protein involved in cell-cell contact formation; NM_(—)017717, MUCDHL, mucin and cadherin-like; NM_(—)005130 HBP17 (heparin-binding growth factor binding protein), which binds to HB-EGF which activates the RAS-signaling pathway; NM_(—)002272, KRT4, Homo sapiens keratin 4 (KRT4), which is involved in adhesion and wound healing; NM_(—)004363 CEACAM5 (Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 5) and NM_(—)002483 CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Carcinoembryonic antigen (CEA) is one of the most frequently used serum tumor markers for carcinoma, particularly in colorectal cancer. The role of CEACAM in tumor growth is complex, with apparently conflicting effects on tumor growth seen in different tumor models. Studies in breast and prostate cancer models suggest that CEACAM may be a tumor suppression gene, whereas other models suggest CEACAM may be involved in tumor invasion and metastasis.

The invention is further illustrated by the following figures, tables and examples.

FIGURE AND TABLE LEGENDS

FIG. 1: Knock down of prohibitin induces changes in epithelial cell morphology. Transfection of siPHB reduces the mRNA level (A) and the protein level (B) of prohibitin compared to control transfections (siLuc or siGFP, respectively). Knock down of prohibitin (siPHB) induces the aggregation and reduced migration of epithelia cancer cells not seen in control cells (siGFP) (C). Transfection of siPHB-3 reduces the PHB mRNA (D) and protein level (E). Expression of the cloned PHB gene pPHB-c in siPHB-3 transfected cells restores PHB expression. PHB-c harbours a N-terminal Flag-tag and has therefore a slightly increased molecular mass compared to endogenous PHB. Expression of the cloned prohibitin pPHB-c in siPHB-3 transfected cells prevents cell cluster formation. For details, see Materials and Methods.

FIG. 2: Knock down of prohibitin induces the formation of adherens junctions and changes in epithelial morphogenesis. Knock down of prohibitin (siPHB) induces the formation of multilayered epithelial cell clusters (A, B) whereas control cells (siLuc) form monolayers (C, D). Samples were analyzed by scanning (SEM, A+C) and transmission electron microscopy (TEM, B+D).

Confocal immunofluorescence analysis of prohibitin knock down cells (E, F, G) and controls (H, I, J). Cells with reduced prohibitin expression show a strong membrane staining for pan-cadherin (E) and β-catenin (F) while staining of control cells reveals a reduced membrane signal and a diffuse cytoplasmic pattern (H, I). G and J show the respective overlays including a DNA stain. G′, J′ and G″,J″ are XZ and YZ reconstructions of confocal Z stacks. The reconstructions clearly reveal that the cell clusters of prohibitin knock down cells are multilayered. Induction of cell migration on collagen by EGF is impaired in prohibitin knock down cells. Shown are the individual frames from various time points of a time-lapse video on the control and prohibitin knock down cells with EGF.

FIG. 3:

Prohibitin is required during EGF induced Raf-MAPK activation Hela cells transfected with control siRNA (siLam), siHer-2 and siPHB were treated with EGF and the phosphorylation of ERK, AKT (A) and Raf-1 (B) was determined by immunoblot analysis with phospho-specific antibodies. Complementation of siPHB transfected HeLa cells restores Raf-1 activation (D). Shown are the levels of Raf-1 pS259 and Raf-1 pS338 blots revealing an increase in Raf-1 pS259 phosphorylation in cells transfected with siPHB-3 (D).

Addition of Phorbol ester (PMA) leads to rapid resolution of cell clusters and the activation of ERK irrespective of PHB levels (E, F). Shown are different time points from a time lapse microscopy of siPHB transfected cells treated with PMA (E). Co-expression of constitutively active RafBXB (G, H) or the cloned PHB gene restore ERK activation and cell cluster formation in siPHB transfected cells.

FIG. 4:

PHB is required for the activation of Raf-1 by Ras.

Prohibitin and Raf-1 were found in the caveolin rich fraction of the plasma membrane in cells with normal prohibitin expression (A; siLuc). Raf-1 is lost from the caveolae in cells with reduced levels of PHB (A; siPHB). The different fractions and the respective concentration of sucrose obtained after gradient centrifugation are indicated. Caveolin-1 (Cav-1) as marker for caveolae as well as PHB and Raf-1 were detected by immuno blotting. Ras-Raf-1 interaction is impaired in PHB knock down cells (B). HeLa cells transfected with control siRNA (siLuc) or siPHB were treated with or without EGF. Ras was then immunoprecipitated using pan-Ras antibody and co-precipitating Raf-1 was detected by immunoblotting (IP). Amounts of PHB and Raf-1 were verified in the whole cell lysates (lysate). Active Ras fails to activate Raf-1 and ERK in PHB knock down cells (D). HeLa cells transfected with control siRNA (siLuc) or siPHB and expression plasmids for H-Ras or H-Ras(G12V) were treated with EGF or buffer. Activation of Raf-1 and ERK was tested by immunoblot analysis using phoshpo-specific antibodies as indicated. Expression of Ras constructs was verified by using anti-HA tag antibody.

FIG. 5:

Nucleotide Sequence encoding PHB (GenBank-Accession-No. NM_(—)002634, SEQ.ID.NO:1). The coding region is given in bold letters.

FIG. 6:

Amino acid sequence of PHB (GenBank-Accession-No. NM_(—)002634, SEQ.ID.NO:2).

FIG. S1: Formation of cell clusters as consequence of silencing PHB expression in HEp-2 cells. Transfection of siPHB-2 reduces the mRNA level (A) and the protein level (B) of prohibitin compared to control transfections (siLuc or siGFP, respectively). Knock down of prohibitin (siPHB-2) induces the aggregation and multilayered organisation of HEp-2 cells not seen in control cells (siGFP) (C).

FIG. S2: Knock down of Her-2 results in the formation of cell clusters. Validation of siHer-2 by quantitative realtime PCR indicates reduced Her-2 mRNA levels of about 80% of control (siLuc) (A). Transfection of siHer-2 induces the formation of cell clusters (B). Transfection of siHer-2 but not of siPHB reduces the surface exposure of Her-2 (C). Shown is a FACS analysis of cells transfected with siHer-2 (No. 1), siPHB (No. 3) or siLuc (No. 4). Cells were stained with anti-Her-2 antibody (Nos. 1, 3, 4) or a isotype control (No. 2). Relative expression of prohibitin, Her-2 and EGFR in cells transfected with siLuc (Luci) or siPHB (PHB) (D).

FIG. S3: Endogenous Raf-1 interacts with PHB

Endogenous PHB was precipitated from EGF or buffer treated HeLa cells and co-precipitating Raf-1 was determined. Raf-1 and Raf-1 pS338 strongly interact with PHB. IP with HA-antibody was used as an isotype control.

Table 1: Genes up-regulated in prohibitin depleted cells

Table 2: Genes down-regulated in prohibitin depleted cells

EXAMPLE 1

Materials and Methods

Cell Culture

HeLa cells, HEp-2 cells, AGS cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (Gibco BRL, Karlsruhe, Germany), and penicillin (100 U/ml)/streptomycin (100 pg/ml) (Gibco BRL, Karlsruhe, Germany) at 37° C. in 5.0% CO₂. Cells were serum starved for 4-6 h prior to the addition of EGF (Promocell) at a final concentration of 20 ng/ml. MEK-1 inhibitor PD98059 was purchased from (Calbiochem).

Transfection of siRNAs

In order to silence expression of prohibitin, 50,000 cells/well were seeded in a 12 well plate at least 20 h prior to transfection. siRNAs were transfected using the Transmessenger transfection kit and RNAifect transfection kit (Qiagen). Two days post transfection, the nearly confluent cells were trypsinized and one half of the cells was seeded on glass cover slips in a 12 well plate for immunofluorescence analysis while the other half was used for western blot analysis. The following siRNAs were employed in this study:

siGFP 5′-AAGUUCAGCGUGUCCGGCGAG-3′, (SEQ.ID.NO:3) siLuc- 5′-AACUUACGCUGAGUACUUCGA-3′, (SEQ.ID.NO:4) siPHB 5′UGUCAACAUCACACUGCGCdTdT3′ (SEQ.ID.NO:5) and GCGCAGUGUGAUGUUGACAdTdT (SEQ.ID.NO:6) siPHB-2 AGCCAGCTTCCTCGCATCTdTdT, (SEQ.ID.NO:7) and AGATGCGAGGAAGCTGGCTdGdG, (SEQ.ID.NO:8) siPHB-3 5′-CCCAGAAAUCACUGUGAAAdTdT-3′, (SEQ.ID.NO:9) and TTTCACAGUGAUUUCUGGGdTdT. (SEQ.ID.NO:10)

For complementation experiments, cells grown to 80% confluence were transfected with the siPHB-3 siRNA (80 nM) and the full length PHB open reading frame cloned in pCDNA3-myc vector (0.5 μg) or the RafBxB construct (0.5 μg) (a kind gift from Ulf Rapp) using RNAifect transfection kit as mentioned before. The control cells were transfected with the empty vector and a siRNA directed against luciferase (siLuc).

Validation of mRNA Levels by Quantitative Realtime PCR

20,000 cells/well were seeded in a 96 well plate one day prior to transfection. Transfection was performed with 0.25 μg siRNA directed against PHB and Luciferase as control and 2 μl Transmessenger per well according to manufacturer's instructions. After 48 h, RNA was isolated using the RNeasy® 96 BioRobot® 8000 system (Qiagen). The relative amount of PHB mRNA was determined by real time PCR using Quantitect™ SYBR® Green RT-PCR Kit from Qiagen following manufacturer's instructions. The expression level of PHB mRNA was normalized against the internal standard GAPDH. The following primers were used:

PHB-5′: 5′-CTTTGACTGCCGTTCTCGAC-3′, (SEQ.ID.NO:11) PHB-3′: 5′-TGGGTGGATTAGTTCTCCAGC-3′, (SEQ.ID.NO:12) and GAPDH-5′: 5′-GGTATCGTGGAAGGACTCATGAC-3′, (SEQ.ID.NO:13) GAPDH-3′: 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′. (SEQ.ID.NO:14)

Preparation of Caveolae Rich Fractions

Detergent extraction and floatation were performed as described previously (22). Shortly, ME-180 or HeLa cells transfected with siRNAs were solubilized in 1% Triton X-100, MBS [Mes-buffered saline; 0.25 M NaCl and 25 mM Mes (pH 6.8)] and a cocktail of protease inhibitors on ice for 1 h without agitation. The cell lysates were adjusted to 45% sucrose in MBS, overlaid with 7 ml of 35% and 2 ml 5% sucrose in MBS, and centrifuged for 18 h at 36,000 rpm in a SW40Ti rotor (Beckman Instruments). Twelve 1 ml fractions were collected from the top of the gradient and were assayed for protein content, caveolin-1, Raf-1, Ras and prohibitin.

SDS-PAGE and Western Blot

For SDS-PAGE, cells were lysed in single detergent buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 μgml⁻¹ Aprotinin, 0.5 μgml⁻¹ Leupeptin, 1 mM Pefabloc, 10 μM Pepstatin) for 15-20 min on ice and sonicated twice for 15 sec. Lysates were cleared by centrifugation for 10 min at 13,000 rpm, Sample buffer was added to these lysates and boiled at 90° C. for 2 min before loading onto the gels. After separation the proteins were transferred to PVDF-membranes. For immunoblot analysis membranes were blocked with 3% BSA in TBS with 0.5% Tween-20 for 2 h and incubated with anti-prohibitin (Neomarkers), anti-alpha tubulin antibody (Sigma), anti-E-cadherin (Zymed labs), anti-Pan-cadherin (Sigma) or anti-beta-catenin (Sigma) anti-caveolin (N20, Santa Cruz Biotechnology), c-Raf rabbit polyclonal antibody (Santa Cruz Biotechnology), c-Raf mouse monoclonal antibody (Pharmingen), Caveolin mouse monoclonal antibody, anti-Raf-1 pS338 and anti-Raf-1 pS259 rabbit monoclonal antibody (Cell signalling), phospho-MEK (Cell signalling), anti-beta actin monoclonal antibody (Sigma), anti-Her-2 rabbit polyclonal antibody from. (Cell signalling) (anti-pan-Ras monoclonal antibody (Pharmingen), phosphor-ERK, Anti-ERK2 antibody, EGFR-antibody and anti-Myc 9E10 mouse monoclonal antibody (Santa Cruz). Antigen antibody complexes were detected by horseradish peroxidase coupled antibodies (Pharmingen) followed by enhanced chemiluminescence (NEN).

FACS Analysis

For surface staining of Her-2, Hela cells transfected with siRNAs were detached by A cutase treatment 60 h post transfection. The cells were washed once with PBS and incubated in PBS with 2% BSA for 20 min at room temperature. The cells were once again washed with PBS and stained with FITC coupled anti-Her-2 antibody (Bender med systems) diluted in BSA containing PBS at a final dilution of 1:50 and incubated at room temperature for 40 min. At the end of the incubation the cells were washed twice with PBS and the labelled cells were detected using a Becton Dickinson flow cytometer.

Immunoprecipitation

Cells were washed in ice cold PBS and lysed in RIPA buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 1 mM NaVO₃, 10 mM Na-pyrophosphate, 1 mM NaF, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1 μg/ml Aprotinin, 0.5 μg/ml Leupeptin, 1 mM Pefabloc, 10 μM Pepstatin for 30 min on ice and sonicated twice for 10 sec. Lysates were cleared by centrifugation for 15 min at 13,000 rpm. Supernatants were incubated with agarose-coupled anti-H-Ras (Santa Cruz Biotechnology) or Raf-1 (C-12, Santa Cruz Biotechnology) for over night. In the latter case the antigen-antibody complexes were pull down by Sepharose coupled protein A/G beads.

In Vitro Kinase Assay

For in vitro kinase assays, Hela cells transfected with siRNAs were treated with or without EGF and cell lysates were prepared for immunoprecipitations as mentioned before. The immunoprecipitated Raf-1 was washed twice with RIPA buffer and kinase buffer w/o ATP (25 mM Hepes-NaOH pH 7.5, 5 mM MgCl₂, 4 mM MnCl₂). The beads were incubated at 30° for 30 min in the presence of [γ-³²P] ATP in kinase buffer supplemented with 30 μM ATP with or without 2 μg of MEK-1 full length protein (Santa Cruz) in a total volume of 40 μl. Kinase reactions were terminated by addition of SDS sample buffer and boiling. The proteins were separated by SDS-PAGE and analyzed by autoradiography.

Transmission Electron Microscopy

siRNA transfected Hela cells were trypsinized and seeded on glass coverslips at 48 h post transfection. After 60 h, cells were fixed with 2.5% glutaraldehyde, postfixed with 0.5% osmium tetroxide and contrasted using tannic acid and uranyl acetate. Specimens were dehydrated in a graded ethanol series and embedded in Polybed. Ultrathin sections were analysed in a Leo 906E transmission electron microscope (Leo Oberkochen).

Scanning Electron Microscopy

Samples grown on glass coverslips were fixed in 2.5% glutaraldehyde, dehydrated in a graded ethanol series, critical-point dried and coated with a layer of 2 nm platinum/carbon. Specimens were analysed in a Leo 1550 field emission scanning electron microscope (Leo, Oberkochen).

Immunofluorescence Microscopy

siRNA transfected cells were seeded on glass coverslips at 48 h post transfection and fixed with 4% PFA/PBS at 60 h post transfection. The fixed cells were washed once with PBS and permeabilised with 1% Triton/PBS for 1 min and blocked (1% BSA, 5% NGS, 0.05% Tween 20 in PBS for 1 h. The samples were incubated with antibodies against Pan-Cadherin (Sigma) and β-Catenin (Sigma) in blocking buffer. After washing, bound antibodies were detected using goat anti-mouse Cy2 and goat anti rabbit Cy3 secondary antibodies. Nuclei were stained with Draq 5 (Bilostatus LTD). Tri colour Z-stacks were generated using a Leica TCS-SP confocal microscope (Leica Microsystems). For 3-dimensional analysis, stacks were processed using Volocity software (Improvision).

EXAMPLE 2

Microarray Method

In a microarray analysis, target genes were identified which are downregulated or upregulated in prohibitin depleted cells. Results are summarized in Tables 1 and 2.

Microarray experiments were carried out as two-color dye-reversal ratio hybridizations on a 44.000 Whole Human Genome Oligo Microarray AMADID 012391 with 37327 records and 43931 features (Agilent Technologies, Palo Alto, Calif., USA). RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 μg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent anti-sense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled anti-sense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A_(552 nm) for Cy3-CTP and A_(650 nm) for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 μg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60° C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 μm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data (G. A. Churchill, Fundamentals of experimental design for cDNA microarrays, Nat Genet 32 Suppl (2002) 490-495), a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Ratio profiles were generated from raw scan data by a processing pipeline which includes pre-processing (Feature Extraction) and post-processing (Rosetta Resolver) of data and error model adjustments to the raw scan data. Ratio profiles were combined in an error-weighted fashion (Rosetta Resolver) to create ratio experiments, and ratio experiments consisted of one or more ratio profiles. Expression patterns were identified using stringent analysis criteria of 2-fold expression cut-offs of the ratio experiments and an anti-correlation of the dye reversal ratio profiles. Anti-correlation was determined by using the ‘compare function’ to match two color-swap dye-reversal hybridizations and to decide how similar or dissimilar they were. In this way, only anti-correlated spots that had on the one array a red colour and on the other one a green colour and vice versa were selected. We compared color-swap dye-reversal hybridizations of individual 2 two-channel hybridizations resulting in unchanged genes, query signature genes, target signature genes, common signature genes and anti-correlated genes. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value<0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system.

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TABLE 1 Genes up-regulated in prohibitin depleted cells Fold Change Accession # Sequence Name(s) Sequence Description 4.91215 NM_032160 NM_032160 Homo sapiens NCAG1 (NCAG1), mRNA 4.39232 BC016329 BC016329 Homo sapiens, clone IMAGE: 4103364, mRNA 3.49996 NM_017831 FLJ20456 hypothetical protein FLJ20456 3.41678 NM_004038 AMY1A amylase, alpha 1A; salivary 3.35519 U49436 EIF5 eukaryotic translation initiation factor 5 3.34253 NM_018038 FLJ10246 hypothetical protein FLJ10246 3.12768 A_32_P160186 A_32_P160186 Unknown 3.10957 AF445027 AF445027 Homo sapiens clone 114 tumor rejection antigen mRNA, complete cds 3.04008 NM_018357 FLJ11196 hypothetical protein FLJ11196 3.03416 NM_017638 FLJ20045 hypothetical protein FLJ20045 2.97146 NM_004923 MTL5 metallothioneln-like 5, testis-specific (tesmin) 2.94765 NM_007193 ANXA10 annexin A10 2.92751 THC1969492 THC1969492 Unknown 2.91389 THC1847341 THC1847341 Unknown 2.89299 NM_001195 BFSP1 beaded filament structural protein 1, filensin 2.83127 NM_020428 CTL2 CTL2 gene 2.78717 NM_012385 P8 p8 protein (candidate of metastasis 1) 2.64887 BC021677 BC021677 Homo sapiens, clone IMAGE: 4045663, mRNA 2.62683 AK095791 AK095791 Homo sapiens cDNA FLJ38472 fis, clone FEBRA2022148 2.61456 NM_020801 ARRDC3 Homo sapiens arrestin domain containing 3 (ARRDC3), mRNA 2.57308 NM_032229 SLITRK6 Homo sapiens SLIT and NTRK-like family, member 6 (SLITRK6), mRNA 2.56148 AK024085 FLJ20069 hypothetical protein FLJ20069 2.51701 NM_005572 LMNA lamin A/C 2.51405 THC1919609 THC1919609 Unknown 2.50532 AK098124 AK098124 Homo sapiens cDNA FLJ40805 fis, clone TRACH2009060 2.4728 NM_000426 LAMA2 laminin, alpha 2 (merosin, congenital muscular dystrophy) 2.45336 AK057267 AK057267 Homo sapiens cDNA FLJ32705 fis, clone TESTI2000600, weakly similar to RESTIN 2.44739 NM_133646 NM_133646 Homo sapiens sterile alpha motif and leucine zipper containing kinase AZK (ZAK), mRNA 2.38943 ENST00000333505 ENST00000333505 Unknown 2.38742 NM_005269 GLI glioma-associated oncogene homolog (zinc finger protein) 2.37759 AL117598 AL117598 Homo sapiens mRNA; cDNA DKFZp564H1663 (from clone DKFZp564H1663) 2.35368 AL832786 AL832786 Homo sapiens mRNA; cDNA DKFZp667D2123 (from clone DKFZp667D2123) 2.3507 NM_004083 DDIT3 DNA-damage-inducible transcript 3 2.34769 AK002152 STAU2 staufen (Drosophila, RNA-binding protein) homolog 2 2.34765 BX647764 BX647764 Homo sapiens mRNA; cDNA DKFZp686E0352 (from clone DKFZp686E0352) 2.33447 BC032558 BC032558 Homo sapiens distal-less homeo box 2, mRNA (cDNA clone MGC: 45154 IMAGE: 5562689), complete cds 2.31817 THC1924048 THC1924048 Unknown 2.28953 NM_017651 FLJ20069 hypothetical protein FLJ20069 2.28536 NM_001326 CSTF3 cleavage stimulation factor, 3′ pre-RNA, subunit 3, 77 kD 2.28313 THC1923482 THC1923482 Unknown 2.25252 NM_024699 NM_024699 Homo sapiens hypothetical protein FLJ14007 (FLJ14007), mRNA 2.24907 A_24_P913835 A_24_P913835 Unknown 2.24753 NM_016447 LOC51678 MAGUK protein p55T; Protein Associated with Lins 2 2.23333 NM_001172 ARG2 arginase, type II 2.22362 NM_013448 BAZ1A bromodomain adjacent to zinc finger domain, 1A 2.21632 BX640843 BX640843 Homo sapiens mRNA; cDNA DKFZp686B14224 (from clone DKFZp686B14224) 2.21413 THC1839547 THC1839547 Unknown 2.21208 NM_003467 CXCR4 chemokine (C-X-C motif), receptor 4 (fusin) 2.2102 NM_152486 NM_152486 Homo sapiens hypothetical protein MGC45873 (MGC45873), mRNA 2.20894 THC1957808 THC1957808 Unknown 2.20499 THC1808814 THC1808814 Unknown 2.20266 NM_004405 DLX2 distal-less homeo box 2 2.18355 AK023854 AK023854 Homo sapiens cDNA FLJ13792 fis, clone THYRO1000072, weakly similar to MYOSIN LIGHT CHAIN KINASE, SMOOTH MUSCLE AND NON-MUSCLE ISOZYMES (EC 2.7.1.117) 2.17592 AK075052 AK075052 Homo sapiens cDNA FLJ90571 fis, clone OVARC1001725, highly similar to Homo sapiens patched related protein TRC8 (TRC8) gene 2.17336 AF001540 CS-1 calcineurin-binding protein calsarcin-1 2.17033 NM_007361 NID2 nidogen 2 2.16647 A_32_P115707 A_32_P115707 Unknown 2.16614 NM_006756 TCEA1 transcription elongation factor A (SII), 1 2.16487 NM_152346 NM_152346 Homo sapiens hypothetical protein MGC34680 (MGC34680), mRNA 2.16351 BC062599 BC062599 Homo sapiens cDNA clone MGC: 74441 IMAGE: 5248209, complete cds 2.15172 BC015814 BC015814 Homo sapiens KIAA1799 protein, mRNA (cDNA clone MGC: 9635 IMAGE: 3915942), complete cds 2.15113 AL832398 AL832398 Homo sapiens mRNA; cDNA DKFZp667M2212 (from clone DKFZp667M2212) 2.1504 NM_148169 FBXO26 Homo sapiens F-box only protein 26 (FBXO26), transcript variant 1, mRNA 2.14271 AL832820 AL832820 Homo sapiens mRNA; cDNA DKFZp667D0824 (from clone DKFZp667D0824) 2.13359 BC042535 BC042535 Homo sapiens F-box only protein 33, mRNA (cDNA clone IMAGE: 4751694), partial cds 2.1333 AK026078 AK026078 Homo sapiens cDNA; FLJ22425 fis, clone HRC08686 2.13303 BC062758 BC062758 Homo sapiens cDNA clone IMAGE: 4081583, partial cds 2.13231 NM_030899 ZNF323 Homo sapiens zinc finger protein 323 (ZNF323), mRNA 2.12847 AK000939 AK000939 Homo sapiens cDNA FLJ10077 fis, clone HEMBA1001864 2.1237 NM_024332 NM_024332 Homo sapiens c6.1A (C6.1A), mRNA 2.1212 BC038180 BC038180 Homo sapiens O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase), transcript variant 1, mRNA (cDNA clone MGC: 39117 IMAGE: 5017795), complete cds 2.12077 AF143879 AF143879 Homo sapiens clone IMAGE: 120631 mRNA sequence. 2.12068 NM_012446 SSBP2 single-stranded-DNA-binding protein 2.11775 AK097639 AK097639 Homo sapiens cDNA FLJ40320 fis, clone TESTI2030770 2.11499 AL110141 AL110141 Homo sapiens mRNA; cDNA DKFZp564D0164 (from clone DKFZp564D0164). 2.1033 NM_018725 IL17BR IL-17B receptor 2.09966 NM_181711 GRASP Homo sapiens GRP1 (general receptor for phosphoinositides 1)-associated scaffold protein (GRASP), mRNA 2.09541 NM_012258 HEY1 hairy/enhancer-of-split related with YRPW motif 1 2.09496 NM_021204 MASA Homo sapiens E-1 enzyme (MASA), mRNA. 2.09345 AW511222 AW511222 ESTs, Moderately similar to 2.08172 NM_006759 UGP2 UDP-glucose pyrophosphorylase 2 2.07536 NM_004734 DCAMKL1 doublecortin and CaM kinase-like 1 2.06927 NM_017996 FLJ10103 hypothetical protein FLJ10103 2.05569 NM_000538 RFXAP regulatory factor X-associated protein 2.05446 THC1871361 THC1871361 Unknown 2.05396 A_24_P516728 A_24_P516728 Unknown 2.04781 A_24_P545200 A_24_P545200 Unknown 2.04682 NM_018698 P15-2 hypothetical protein P15-2 2.03828 NM_138433 NM_138433 Homo sapiens hypothetical protein BC009980 (LOC113730), mRNA 2.03462 NM_001969 EIF5 eukaryotic translation initiation factor 5 2.03183 NM_183013 CREM Homo sapiens cAMP responsive element modulator (CREM), transcript variant 19, mRNA 2.02975 NM_024786 ZDHHC11 Homo sapiens zinc finger, DHHC domain containing 11 (ZDHHC11), mRNA 2.02721 BC026998 BC026998 Homo sapiens, Similar to envoplakin, clone IMAGE: 5107188, mRNA 2.02703 NM_003084 SNAPC3 small nuclear RNA activating complex, polypeptide 3, 50 kD 2.01497 A_32_P118522 A_32_P118522 Unknown 2.01111 THC2005339 THC2005339 Unknown 2.0072 BC030611 BC030611 Homo sapiens cDNA clone MGC: 27088 IMAGE: 4827474, complete cds 2.00265 AF026526 AF026526 Homo sapiens unknown mRNA downregulated by Induced differentiation with 13-cis retinoic acid.

TABLE 2 Genes down-regulated in prohibitin depleted cells Fold Change Accession # Sequence Name(s) Sequence Description −2.00202 NM_001935 DPP4 dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing protein 2) −2.00298 AL080111 AL080111 Homo sapiens mRNA; cDNA DKFZp586G2222 (from clone DKFZp586G2222) −2.00413 THC1971019 THC1971019 Unknown −2.00532 BC036187 BC036187 Homo sapiens serine/arginine repetitive matrix 1, mRNA (cDNA clone MGC: 39488 IMAGE: 4827738), complete cds −2.00654 NM_000873 ICAM2 Intercellular adhesion molecule 2 −2.00705 U17077 BENE BENE protein −2.00718 AK027134 AK027134 Homo sapiens cDNA: FLJ23481 fis, clone KAIA03003. −2.00915 THC1960903 THC1960903 Unknown −2.01233 BC026020 BC026020 Homo sapiens, Similar to KIAA0562 gene product, clone IMAGE: 4875334, mRNA −2.01781 BC029358 BC029358 Homo sapiens G protein-coupled receptor 110, mRNA (cDNA clone IMAGE: 4812868), containing frame-shift errors −2.01879 X97261 MT1L metallothioneln 1L −2.01979 A_32_P99533 A_32_P99533 Unknown −2.02275 NM_014889 MP1 metalloprotease 1 (pitrilysin family) −2.0241 NM_005186 CAPN1 Homo sapiens calpain 1, (mu/l) large subunit (CAPN1), mRNA. −2.02523 NM_030812 NM_030812 Homo sapiens actin like protein (LOC81569), mRNA −2.02584 NM_021180 NM_021180 Homo sapiens sister-of-mammalian grainyhead (SOM), transcript variant 1, mRNA −2.02612 THC1957838 THC1957838 Unknown −2.02672 NM_001144 AMFR autocrine motility factor receptor −2.02765 NM_016229 LOC51700 cytochrome b5 reductase b5R.2 −2.02802 AK055065 AK055065 Homo sapiens cDNA FLJ30503 fis, clone BRAWH2000494 −2.0296 A_24_P118361 A_24_P118361 Unknown −2.02984 BC004277 BC004277 Homo sapiens cDNA clone MGC: 10837 IMAGE: 3615489, complete cds −2.03347 ENST00000330567 ENST00000330567 Unknown −2.03436 THC1910374 THC1910374 Unknown −2.03465 BC007117 BC007117 Homo sapiens, clone IMAGE: 4333276, mRNA, partial cds −2.0355 THC1940812 THC1940812 Unknown −2.0366 AK074229 AK074229 Homo sapiens cDNA FLJ23649 fis, clone COL06395, highly similar to Oryctolagus cuniculus lacrimal lipase mRNA −2.03806 NM_000346 SOX9 SRY (sex-determining region Y)-box 9 (campomelic dysplasia, autosomal sex-reversal) −2.03887 ENST00000330875 ENST00000330875 Unknown −2.04904 NM_002612 PDK4 pyruvate dehydrogenase kinase, isoenzyme 4 −2.05042 BC012091 BC012091 Homo sapiens hairy and enhancer of split (Drosophila) homolog 2, mRNA (cDNA clone MGC: 20048 IMAGE: 4834002), complete cds −2.05083 THC1958676 THC1958676 Unknown −2.05132 A_24_P247303 A_24_P247303 Unknown −2.05232 A_24_P230466 A_24_P230466 Unknown −2.05305 NM_080430 NM_080430 Homo sapiens selenoprotein M (SELM), mRNA −2.05578 NM_032012 C9orf5 Homo sapiens chromosome 9 open reading frame 5 (C9orf5), mRNA −2.05599 NM_000696 ALDH9 aldehyde dehydrogenase 9 (gamma-aminobutyraldehyde dehydrogenase, E3 isozyme) −2.05617 A_24_P247233 A_24_P247233 Unknown −2.05929 NM_006108 SPON1 Homo sapiens spondin 1, extracellular matrix protein (SPON1), mRNA −2.06232 NM_014256 TMEM3 transmembrane protein 3 −2.06567 A_24_P792988 A_24_P792988 Unknown −2.06675 NM_199293 TH Homo sapiens tyrosine hydroxylase (TH), transcript variant 3, mRNA −2.06697 NM_025080 ASRGL1 Homo sapiens asparaginase like 1 (ASRGL1), mRNA −2.06789 THC1991570 THC1991570 Unknown −2.06805 AK024513 AK024513 Homo sapiens cDNA; FLJ20860 fis, clone ADKA01632. −2.06874 THC1881737 THC1881737 Unknown −2.06884 A_24_P358857 A_24_P358857 Unknown −2.07022 AK092260 AK092260 Homo sapiens cDNA FLJ34941 fis, clone NT2RP7007480 −2.07198 AK075362 AK075362 Homo sapiens cDNA PSEC0048 fis, clone NT2RP2000028, highly similar to Homo sapiens serine protease mRNA −2.07486 A_34_P471242 A_24_P471242 Unknown −2.0763 NM_018584 PRO1489 hypothetical protein PRO1489 −2.07713 ENST00000333197 ENST00000333197 Unknown −2.07923 NM_004360 CDH1 cadherin 1, type 1, E-cadherin (epithelial) −2.07957 AK002195 AK002195 Homo sapiens cDNA FLJ11333 fis, clone PLACE1010616 −2.08301 A_24_P530977 A_24_P530977 Unknown −2.08542 ENST00000327707 ENST00000327707 Unknown −2.08675 A_24_P186746 A_24_P186746 Unknown −2.09026 NM_002230 JUP Junction piakogiobin −2.09252 NM_000693 ALDH6 aldehyde dehydrogenase 6 −2.09611 THC1881942 THC1881942 Unknown −2.09756 NM_138768 MYEOV Homo sapiens myeloma overexpressed gene (In a subset of t(11; 14) positive multiple myelomas) (MYEOV), mRNA −2.09924 NM_007267 LAK-4P expressed in activated T/LAK lymphocytes −2.10043 AK024480 FLJ00074 Homo sapiens mRNA for FLJ00074 protein, partial cds −2.103 AK022268 AK022268 Homo sapiens cDNA FLJ12206 fis, clone MAMMA1000941 −2.10499 NM_018192 FLJ10718 hypothetical protein FLJ10718 −2.10701 A_24_P84970 A_24_P84970 Unknown −2.1111 NM_000112 SLC26A2 solute carrier family 26 (sulfate transporter), member 2 −2.11277 NM_014292 CBX6 chromobox homolog 6 −2.11301 A_24_P109661 A_24_P109661 Unknown −2.11559 NM_004753 SDR1 short-chain dehydrogenase/reductase 1 −2.12023 A_24_P375360 A_24_P375360 Unknown −2.12417 BC036649 BC036649 Homo sapiens Sec23 homolog A (S. cerevisiae), mRNA (cDNA clone MGC: 26267 IMAGE: 4821858), complete cds −2.12449 BC007034 BC007034 Homo sapiens metallothionein 2A, mRNA (cDNA clone MGC: 12397 IMAGE: 4051220), complete cds −2.12555 AK021884 NPEPPS aminopeptidase puromycin sensitive −2.13148 A_24_P350136 A_24_P350136 Unknown −2.13248 NM_020402 CHRNA10 cholinergic receptor, nicotinic, alpha polypeptide 10 −2.13329 AF161448 HSPC016 hypothetical protein −2.13427 AL833323 AL833323 Homo sapiens mRNA; cDNA DKFZp686G1128 (from clone DKFZp686G1128) −2.13627 AK095472 AK095472 Homo sapiens cDNA FLJ38153 fis, clone DFNES1000083 −2.13814 NM_005737 ARL7 ADP-ribosylation factor-like 7 −2.14183 A_24_P780319 A_24_P780319 Unknown −2.14398 NM_025214 NM_025214 Homo sapiens CTCL tumor antigen se57-1 (SE57-1), mRNA −2.14628 A_32_P80809 A_32_P80809 Unknown −2.14944 BC002759 BC002759 Homo sapiens hypothetical protein FLJ20489, mRNA (cDNA clone IMAGE: 3632656), complete cds −2.14967 BC049193 BC049193 Homo sapiens KIAA1277, mRNA (cDNA clone IMAGE: 4445131), partial cds −2.15143 A_24_P922101 A_24_P922101 Unknown −2.15502 NM_005953 MT2A metallothionein 2A −2.15502 L32537 L32537 Homo sapiens (clone XP6G6B) mRNA, partial EST. −2.1592 A_32_P182135 A_32_P182135 Unknown −2.16029 NM_032409 PINK1 Homo sapiens PTEN induced putative kinase 1 (PINK1), mRNA −2.16178 THC1953717 THC1953717 Unknown −2.1701 THC1940902 THC1940902 Unknown −2.17121 NM_006460 HIS1 HMBA-inducible −2.17172 NM_015894 SCLIP SCG10-like-protein −2.17557 AK096428 AK096428 Homo sapiens cDNA FLJ39109 fis, clone NTONG2005137, highly similar to [PYRUVATE DEHYDROGENASE(LIPOAMIDE)] KINASE ISOZYME 4, MITOCHONDRIAL PRECURSOR (EC 2.7.1.99) −2.17744 AK055851 AK055851 Homo sapiens cDNA FLJ31289 fis, clone KIDNE2007328 −2.17823 NM_004878 PTGES prosteglandin E synthase −2.17957 NM_002276 KRT19 keratin 19 −2.18837 ENST00000332472 ENST00000332472 Unknown −2.19207 AK125106 AK125106 Homo sapiens cDNA FLJ43116 fis, clone CTONG3002127, weakly similar to Mus musculus synaptotagmin-like 4 (Sytl4) −2.1944 THC1993191 THC1993191 Unknown −2.19924 NM_001848 COL6A1 Homo sapiens collagen, type VI, alpha 1 (COL6A1), mRNA −2.19977 BC035496 BC035496 Homo sapiens Cbp/p300-Interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 4, mRNA (cDNA clone MGC: 19748 IMAGE: 3622207), complete cds −2.1999 NM_198473 NM_198473 Homo sapiens FLJ46111 protein (FLJ46111), mRNA −2.20097 AF092095 TIP30 Tat-Interacting protein (30 kD) −2.20981 BC004295 BC004295 Homo sapiens, clone IMAGE: 3622356, mRNA, partial cds −2.21584 THC1952994 THC1952994 Unknown −2.21861 NM_006651 CPLX1 complexin 1 −2.2225 NM_002614 PDZK1 PDZ domain containing 1 −2.22477 X16896 IL1R1 Interleukin 1 receptor, type I −2.22745 THC1846762 THC1846762 Unknown −2.22906 NM_003370 VASP vasodilator-stimulated phosphoprotein −2.23039 ENST00000311197 ENST00000311197 Unknown −2.23181 AK025062 AK025062 Homo sapiens cDNA: FLJ21409 fis, clone COL03924 −2.23286 NM_003500 ACOX2 acyl-Coenzyme A oxidase 2, branched chain −2.24005 BC036885 BC036885 Homo sapiens, clone IMAGE: 5399920, mRNA −2.24334 NM_206827 RASL11A Homo sapiens RAS-like, family 11, member A (RASL11A), mRNA −2.24932 NM_005952 MT1X Homo sapiens metallothionein 1X (MT1X), mRNA. −2.25159 AK027217 LIM LIM protein (similar to rat protein kinase C-binding enigma) −2.25366 AB017116 AD 1 Homo sapiens mitochondrial mRNA for AD 1, partial cds. −2.25688 U06469 SLC1A1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 −2.25716 NM_022369 FLJ12541 Homo sapiens hypothetical protein FLJ12541 similar to Stra6 (FLJ12541), mRNA. −2.26077 A_24_P928250 A_24_P928250 Unknown −2.26084 THC1860502 THC1860502 Unknown −2.26869 NM_138448 ACYP2 Homo sapiens acylphosphatase 2, muscle type (ACYP2), mRNA −2.26924 NM_004695 SLC16A5 solute carrier family 16 (monocarboxylic acid transporters), member 5 −2.26993 A_32_P104469 A_32_P104469 Unknown −2.27435 Z34282 MUC5AC H. sapiens (MAR11) MUC5AC mRNA for mucin (partial) −2.27521 NM_014452 DR6 death receptor 6 −2.28033 A_24_P315594 A_24_P315594 Unknown −2.28449 A_24_P917810 A_24_P917810 Unknown −2.28898 L02922 tropomyosin Human tropomyosin isoform hTM5a mRNA, exon 1. −2.29203 AK091178 AK091178 Homo sapiens cDNA FLJ33859 fis, clone CTONG2006223, moderately similar to KERATIN, TYPE II CYTOSKELETAL 8 −2.2968 ENST00000322533 ENST00000322533 Unknown −2.30396 NM_139314 ANGPTL4 Homo sapiens angiopoietin-like 4 (ANGPTL4), transcript variant 1, mRNA −2.3124 NM_022119 PRSS22 Homo sapiens protease, serine, 26 (PRSS22), mRNA. −2.32359 NM_138967 SCAMP5 Homo sapiens secretory carrier membrane protein 5 (SCAMP5), mRNA −2.32421 NM_017878 FLJ20556 hypothetical protein FLJ20556 −2.32808 NM_002646 PIK3C2B Homo sapiens phosphoinositide-3-kinase, class 2, beta polypeptide (PIK3C2B), mRNA. −2.32922 AK074567 AK074567 Homo sapiens cDNA FLJ90086 fis, clone HEMBA1005145 −2.3301 NM_016201 LCCP Leman coiled-coil protein −2.34004 NM_182494 NM_182494 Homo sapiens hypothetical protein LOC119395 (LOC119395), mRNA −2.34802 BF129169 BF129169 ESTs −2.352 X91221 NCX1 H. sapiens mRNA for NCX1 protein 3′UTR. −2.35349 BC032946 BC032946 Homo sapiens aquaporin 5, mRNA (cDNA clone MGC: 33163 IMAGE: 5269384), complete cds −2.36509 A_24_P843309 A_24_P843309 Unknown −2.3684 NM_020353 LOC57088 phospholipid scramblase 4 −2.37251 NM_017458 MVP major vault protein −2.3833 A_24_P844100 A_24_P844100 Unknown −2.38554 NM_005476 GNE UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase −2.38839 NM_004635 MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 −2.39088 NM_003740 KCNK5 potassium channel, subfamily K, member 5 (TASK-2) −2.39301 NM_031311 CPVL Homo sapiens carboxypeptidase, viteilogenic-like (CPVL), transcript variant 1, mRNA −2.39894 A_32_P152999 A_32_P152999 Unknown −2.40969 NM_000142 FGFR3 fibroblast growth factor receptor 3 (achondroplasia, thanatophoric dwarfism) −2.41275 THC1905270 THC1905270 Unknown −2.41493 THC1944835 THC1944835 Unknown −2.42522 ENST00000328006 ENST00000328006 Unknown −2.42726 NM_002273 KRT8 keratin 8 −2.42838 NM_002305 LGALS1 lectin, galactoside-binding, soluble, 1 (galectin 1) −2.43097 BF512676 BF512676 ESTs −2,43469 A_32_P202708 A_32_P202708 Unknown −2.43629 NM_014471 PEC-60 gastrointestinal peptide −2.43751 NM_001897 CSPG4 chondroltin sulfate proteoglycan 4 (melanoma-associated) −2.43756 NM_003896 SIAT9 sialyltransferase 9 (CMP-NeuAc: lactosylceramide alpha-2,3-sialyltransferase; GM3 synthase) −2.44725 A_24_P306644 A_24_P306844 Unknown −2.45084 BC009792 BC009792 Homo sapiens cleavage stimulation factor, 3′ pre-RNA, subunit 3, 77 kDa, mRNA (cDNA clone IMAGE: 4274695), complete cds −2.4533 BQ362073 BQ362073 Unknown −2.45467 A_24_P15973 A_24_P15973 Unknown −2.45903 BC040110 BC040110 Homo sapiens, Similar to fer-1-like 3, myoferlin (C. elegans), clone IMAGE: 3882757, mRNA, partial cds −2.46008 A_24_P418216 A_24_P418216 Unknown −2.467 NM_170726 ALDH4A1 Homo sapiens aldehyde dehydrogenase 4 family, member A1 (ALDH4A1), nuclear gene encoding mitochondrial protein, transcript variant P5CDhS, mRNA −2.4732 NM_017451 BAIAP2 BAI1-associated protein 2 −2.47787 THC1893261 THC1893261 Unknown −2.47944 NM_003255 TIMP2 tissue Inhibitor of metelloproteinase 2 −2.4888 NM_005368 MB myoglobin −2.50196 NM_001548 IFIT1 Interferon-Induced protein with tetratricopeptide repeats 1 −2.51182 NM_002275 KRT15 keratin 15 −2.51385 NM_005971 FXYD3 FXYD domain-containing Ion transport regulator 3 −2.5213 NM_000716 C4BPB complement component 4-binding protein, beta −2.52213 NM_182741 MUC1 Homo sapiens mucin 1, transmembrane (MUC1), mRNA −2.52676 NM_001549 IFIT4 Interferon-Induced protein with tetratricopeptide repeats 4 −2.5292 NM_016619 LOC51316 hypothetical protein −2.533 AK075564 AK075564 Homo sapiens cDNA PSEC0264 fis, clone NT2RP3002337 −2.53403 A_24_P7021 A_24_P7021 Unknown −2.54393 NM_182532 NM_182532 Homo sapiens hypothetical protein LOC199964 (LOC199964), mRNA −2.54549 A_24_P83111 A_24_P93111 Unknown −2.56068 A_24_P7750 A_24_P7750 Unknown −2.56166 A_24_P281605 A_24_P281605 Unknown −2.57194 BX374774 BX374774 Unknown −2.57249 BC014851 BC014851 Homo sapiens lunatic fringe homolog (Drosophila), mRNA (cDNA clone MGC: 22145 IMAGE: 4453156), complete cds −2.58019 BC040210 BC040210 Homo sapiens, aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase), clone IMAGE: 4825338, mRNA −2.58072 A_24_P478940 A_24_P478940 Unknown −2.59118 AK097804 AK097804 Homo sapiens cDNA FLJ40485 fis, clone TESTI2043857, moderately similar to Homo sapiens nolp mRNA −2.59217 A_24_P213336 A_24_P213336 Unknown −2.59289 A_23_P120545 A_23_P120545 Unknown −2.60575 NM_173481 C19orf21 Homo sapiens chromosome 19 open reading frame 21 (C19orf21), mRNA −2.61387 NM_032943 SYTL2 Homo sapiens synaptotagmin-like 2 (SYTL2), transcript variant a, mRNA −2.62052 AF034803 PPFIBP2 PTPRF interacting protein, binding protein 2 (liprin beta 2) −2.62447 AK074149 AK074149 Homo sapiens mRNA for FLJ00222 protein −2.62707 NM_002966 S100A10 S100 calcium-binding protein A10 (annexin II ligand, calpactin I, light polypeptide (p11)) −2.62858 NM_020792 NM_020792 Homo sapiens KIAA1363 protein (KIAA1363), mRNA −2.6377 A_24_P341546 A_24_P341546 Unknown −2.64372 NM_152353 NM_152353 Homo sapiens hypothetical protein MGC33839 (MGC33839), mRNA −2.64623 NM_013321 SNX8 Homo sapiens sorting nexin 8 (SNX8), mRNA −2.65233 NM_152864 C20orf58 Homo sapiens chromosome 20 open reading frame 58 (C20orf58), mRNA −2.66028 M59979 PTGS1 prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) −2.6608 BC005987 BC005987 Homo sapiens interferon-induced protein with tetratricopeptide repeats 2, mRNA (cDNA clone IMAGE: 3838493), containing frame-shift errors −2.67518 A_24_P195974 A_24_P195974 Unknown −2.67611 NM_004669 CLIC3 chloride intracellular channel 3 −2.69138 Y12878 ncx1 Homo sapiens NCX1 mRNA alternative 5′end, exon 1d, 1c and 2. −2.69364 NM_019894 TMPRSS4 Homo sapiens transmembrane protease, serine 4 (TMPRSS4), transcript variant 1, mRNA −2.69856 NM_004170 SLC1A1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1 −2.70809 X90980 aml1 Homo sapiens mRNA for an acute myeloid leukaemia protein (486 bp). −2.71286 A_32_P112546 A_32_P112546 Unknown −2.71657 NM_013434 CSEN Calsenilin, presenilin-binding protein, EF hand transcription factor −2.7278 A_24_P401124 A_24_P401124 Unknown −2.7392 NM_003739 AKR1C1 aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase) −2.77465 A_32_P75141 A_32_P75141 Unknown −2.81822 AK095421 AK095421 Homo sapiens cDNA FLJ38102 fis, clone D3OST2000618, moderately similar to Drosophila melanogaster slingshot mRNA −2.82133 NM_144587 C10orf87 Homo sapiens chromosome 10 open reading frame 87 (C10orf87), mRNA −2.82251 AB083038 AB083038 Homo sapiens RDH-E2 mRNA for retinal short chain dehydrogenase reductase, complete cds −2.82589 NM_032034 SLC4A11 Homo sapiens solute carrier family 4, sodium bicarbonate transporter-like, member 11 (SLC4A11), mRNA −2.8323 A_24_P247074 A_24_P247074 Unknown −2.84091 U21049 DD96 epitheilal protein up-regulated in carcinoma, membrane associated protein 17 −2.84421 NM_000891 KCNJ2 potassium inwardly-rectifying channel, subfamily J, member 2 −2.85298 NM_001353 AKR1C1 aldo-keto reductase family 1, member C1 (dihydrodiol dehydrogenase 1; 20-alpha (3-alpha)-hydroxysteroid dehydrogenase) −2.86724 NM_031419 NM_031419 Homo sapiens molecule possessing ankyrin repeats induced by lipopolysaccharide (MAIL), homolog of mouse (MAIL), mRNA −2.88219 BC018597 BC018597 Homo sapiens, clone IMAGE: 3869276, mRNA −2.9014 NM_016434 LOC51750 helicase-like protein NHL −2.91156 NM_024625 ZC3HAV1 Homo sapiens zinc finger CCCH type, antiviral 1 (ZC3HAV1), transcript variant 2, mRNA −2.94339 NM_024306 FA2H Homo sapiens fatty acid 2-hydroxylase (FA2H), mRNA −2.94648 AB028955 KIAA1032 KIAA1032 protein −2.95121 NM_002274 KRT13 keratin 13 −2.97481 NM_138632 NM_138632 Homo sapiens Tara-like protein (HRIHFB2122), transcript variant 2, mRNA −2.98332 BC029496 BC029496 Homo sapiens protein tyrosine phosphatase, receptor type, S, mRNA (cDNA clone IMAGE: 5272670), complete cds −3.00058 THC1927382 THC1927382 Unknown −3.0138 NM_006123 IDS iduronate 2-sulfatase (Hunter syndrome) −3.01734 NM_003733 OASL Homo sapiens 2′-5′oligoadenylate synthetase-like (OASL), mRNA. −3.03167 NM_004363 CEACAM5 Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mRNA. −3.05601 NM_003225 TFF1 trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) −3.10811 U64094 IL1R2 interleukin 1 receptor, type II −3.11205 NM_001547 IFIT2 Homo sapiens interferon-induced protein with tetratricopeptide repeats 2 (IFIT2), mRNA −3.12631 NM_004419 DUSP5 dual specificity phosphatase 5 −3.16603 BC007533 BC007533 Homo sapiens neuropilin 1, mRNA (cDNA clone MGC: 15383 IMAGE: 2958475), complete cds −3.17057 NM_004368 CNN2 calponin 2 −3.19544 AJ001402 MUC5AC mucin 5, subtypes A and C, tracheobronchial/gastric −3.22204 BC069216 BC069216 Homo sapiens cDNA clone MGC: 78739 IMAGE: 6376829, complete cds −3.29685 NM_000213 ITGB4 ,integrin, beta 4 −3.24972 NM_005101 ISG15 interferon-stimulated protein, 15 kDa −3.25168 NM_005423 TFF2 trefoil factor 2 (spasmolytic protein 1) −3.25561 U91543 CHD3 chromodomain helicase DNA binding protein 3 −3.30115 AK056142 AK056142 Homo sapiens cDNA FLJ31580 fis, clone NT2RI2002041 −3.33043 AF311286 AF311286 Homo sapiens tissue transglutaminase (TGM2) mRNA, partial cds −3.3437 THC2003438 THC2003438 Unknown −3.3638 NM_003541 H4FD H4 histone family, member D −3.37813 NM_021968 H4FE Homo sapiens H4 histone family, member E (H4FE), mRNA. −3.42093 A_24_P614940 A_24_P614940 Unknown −3.42714 NM_005672 PSCA prostate stem cell antigen −3.48275 NM_000962 PTGS1 Homo sapiens prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) (PTGS1), mRNA. −3.49519 THC1891572 THC1891572 Unknown −3.52709 NM_003225 TFF1 trefoil factor 1 (breast cancer, estrogen-inducible sequence expressed in) −3.54417 A_23_P66347 A_23_P66347 Unknown −3.73647 NM_144777 SCEL Homo sapiens sciellin (SCEL), transcript variant 2, mRNA −3.78901 BC015940 BC015940 Homo sapiens 5′-nucleotidase, ecto (CD73), mRNA (cDNA clone IMAGE: 3920680), complete cds −4.03972 THC1941189 THC1941189 Unknown −4.07695 NM_017717 MUCDHL mucin and cadherin-like −4.09543 NM_148672 CCL28 Homo sapiens chemokine (C-C motif) ligand 28 (CCL28), transcript variant 2, mRNA −4.09875 BC017859 BC017859 Homo sapiens trefoil factor 3 (intestinal), mRNA (cDNA clone MGC: 22588 IMAGE: 4696566), complete cds −4.09911 NM_003226 TFF3 trefoil factor 3 (intestinal) −4.21393 A_32_P115077 A_32_P115077 Unknown −4.21838 NM_018414 SIAT7A Homo sapiens sialyltransferase 7 ((alpha-N-acetylneuraminyl-2,3-beta-galactosyl-1,3)- N-acetyl galactosaminide alpha-2,6-sialyltransferase) A (SIAT7A), mRNA −4.33931 NM_002634 PHB prohibitin −4.34628 A_24_P349606 A_24_P349606 Unknown −4.35457 NM_024508 ZBED2 Homo sapiens zinc finger, BED domain containing 2 (ZBED2), mRNA −4.3942 NM_032689 ZNF607 Unknown −4.39683 A_24_P50639 A_34_P50639 Unknown −4.58843 ENST00000329070 ENST00000329070 Unknown −4.65917 NM_002193 INHBB inhibin, beta B (activin AB beta polypeptide) −4.69951 A_23_P113692 A_23_P113692 Unknown −4.69982 BC014228 BC014228 Homo sapiens cDNA clone MGC: 20874 IMAGE: 4547239, complete cds −4.71106 BC013401 BC013401 Homo sapiens prohibitin, mRNA (cDNA clone MGC: 3926 IMAGE: 3010198), complete cds −4.90254 NM_019601 BK65A6.2 Homo sapiens Sushi domain (SCR repeat) containing (BK65A6.2), mRNA. −4.90852 A_24_P238896 A_24_P238896 Unknown −4.93829 NM_005130 HBP17 heparin-binding growth factor binding protein −5.06624 NM_004633 IL1R2 interleukin 1 receptor, type II −5.08793 A_24_P203984 A_24_P203984 Unknown −5.41703 BC018706 BC018706 Homo sapiens C-terminal tensin-like, mRNA (cDNA clone MGC: 31757 IMAGE: 5013235), complete cds −5.44197 NM_002483 CEACAM6 carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen) −5.52721 NM_002272 KRT4 Homo sapiens keratin 4 (KRT4), mRNA −5.67766 A_24_P152845 A_24_P152845 Unknown −5.71755 ENST00000331608 ENST00000331608 Unknown −5.78911 NM_020299 AKR1B10 Homo sapiens aldo-keto reductase family 1, member B10 (aldose reductase) (AKR1B10), mRNA −7.96627 BC005008 BC005008 Homo sapiens carcinoembryonic antigen-related cell adhesion molecule 6 (non-specific cross reacting antigen), mRNA (cDNA clone MGC: 10467 IMAGE: 3640231), complete cds 

1-38. (canceled)
 39. Use of at least one inhibitor of Prohibitin (PHB) optionally together with pharmaceutically acceptable carriers, adjuvants, diluents or/and additives for the manufacture of a pharmaceutical composition for the treatment or/and prevention of hyperprolerative disorders.
 40. Use as claimed in claim 39, wherein the pharmaceutical composition is for the treatment or/and prevention of tumours, metastatic tumours, benign tumours, carcinoma, neoplastic carcinoma, gastric carcinoma, larynx carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, bladder carcinoma, EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, or/and Raf-1 transformed tumours.
 41. Use as claimed in claim 39, wherein the at least one inhibitor specifically inhibits the interaction of PHB with a Raf kinase, in particular the binding of PHB to a Raf kinase, preferably Raf-1.
 42. Use as claimed in claim 39, wherein the at least one inhibitor inhibits activation of the Ras-Raf signalling pathway, preferably the Ras-Raf-MAPK pathway or/and the PHB dependent branch of the Ras signalling pathway.
 43. Use as claimed in claim 39, wherein the at least one inhibitor specifically disrupts a Ras-Raf interaction.
 44. Use as claimed in claim 39, wherein the at least one inhibitor inhibits downstream of members of the EGFR family, in-2-particular downstream of EGFR and Her-2.
 45. Use as claimed in claim 39, wherein the at least one inhibitor converts tumour cells from a transformed to a non-transformed phenotype at least in vitro and in vivo.
 46. Use as claimed in claim 39, wherein the at least one inhibitor prevents angiogenesis in tumours.
 47. Use as claimed in claim 39, wherein the at least one inhibitor prevents cell migration in vitro or/and in vivo.
 48. Use as claimed in claim 39, wherein the at least one inhibitor downregulates PHB transcription or/and translation, inhibits targeting of PHB to membranes, inhibits targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes, or/and inhibits posttranslational modification of PHB, in particular required for the PHB targeting to membranes or/and required for the targeting of a Raf kinase, in particular Raf-1 (C-Raf) to membranes.
 49. Use as claimed in claim 39, wherein the at least one inhibitor is selected from the group of nucleic acids, nucleic acid analogues such as ribozymes, peptides, polypeptides, and antibodies.
 50. Use as claimed in claim 49, wherein the nucleic acid is (i) an RNA molecule capable of RNA interference, (ii) a precursor of the RNA molecule (i), or (iii) a DNA molecule encoding the RNA molecule (i) or the precursor (ii).
 51. Use as claimed in claim 50, wherein the DNA molecule is a vector.
 52. Use as claimed in claim 49, wherein the RNA molecule is a double-stranded RNA molecule, preferably a double-stranded siRNA molecule with or without a single-stranded overhang alone at one end or at both ends.
 53. Use as claimed in claim 49, wherein the nucleic acid is an antisense nucleic acid.
 54. Use as claimed in claim 49, wherein the peptide or/and the polypeptide is biologically inactive, capable of inhibiting the activity of PHB, and comprises a fragment of SEQ.ID.NO:2, a derivative thereof, a fragment to of a Raf kinase or/and a derivative thereof.
 55. Use as claimed in claim 49, wherein the antibody is directed against PHB.
 56. Use as claimed in claim 55, wherein the antibody is directed against a peptide or polypeptide comprising (a) the amino acid sequence of SEQ.ID.NO:2, (b) an amino acid sequence which is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence of (a), or/and (c) an immunogenic fragment of (a) or (b).
 57. A nucleic acid suitable as inhibitor in a pharmaceutical composition as defined in claim 39 comprising (a) a fragment of the nucleotide sequence of SEQ.ID.NO:1, or (b) a fragment which is at least 70%, preferably at least 80%, more preferably at least 90% homologous to the sequence of (a).
 58. The nucleic acid as claimed in claim 57 having a length of at least 15, preferably at least 17, more preferably at least 19, most preferably at least 21 nucleotides.
 59. The nucleic acid as claimed in claim 57, which has a length of at the maximum 29, preferably at the maximum 27, more preferably at the maximum 25, especially more preferably at the maximum 23, most preferably at the maximum 21 nucleotides.
 60. The nucleic acid as claimed in claim 57, which is an RNA molecule, preferably a double-stranded RNA molecule, more preferably a double-stranded siRNA molecule with or without a single-stranded overhang alone at one end or at both ends.
 61. The nucleic acid as claimed in 57 comprising a sequence selected from the group consisting of SEQ.ID.NO:5, SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID,NO:8, SEQ.ID.NO;9, SEQ,ID.NO:10 and fragments thereof which may have a length of at least 15 nucleotides.
 62. A screening method for identification of a compound suitable as inhibitor in a pharmaceutical composition defined in claim 39, comprising the steps (a) providing a cell or/and a transgenic non-human animal capable of expressing, particularly overexpressing PHB, (b) contacting a compound with the cell or/and the transgenic non-human animal, (c) determining the amount or/and the activity of PHB, and (d) selecting a compound which reduces the amount or/and the activity of PHB.
 63. The screening method as claimed in claim 62, wherein the activity of PHB in steps (c) and (d) is determined by measuring PHB expression, by prevention of cell migration, by determining targeting of PHB to membranes, by determining targeting of a Raf kinase, in particular Raf-1 to membranes, or/and by determining posttranslational modification of PHB.
 64. The screening method as claimed in claim 62, wherein the activity of PHB in steps (c) and (d) is an interaction of PHB with a Raf kinase, in particular the binding of PHB to a Raf kinase, preferably Raf-1, or/and targeting of a Raf kinase, preferably Raf-1, to membranes.
 65. A screening method for identification of a compound suitable as inhibitor in a pharmaceutical composition defined in claim 62, comprising the steps (a) contacting a compound with isolated PHB or/and an isolated Raf kinase, (b) determining the activity of PHB to interact with Raf kinase, and (c) selecting a compound which reduces the activity of PHB.
 66. The screening method as claimed in claim 65, wherein the interaction is the binding of PHB to a Raf kinase, preferably Ref-1 or/and targeting of a Raf kinase, preferably Raf-1, to membranes.
 67. A method for treating or/and prevention of hyperprolerative disorders comprising the administration of a pharmaceutical composition of claim 39 to a subject in need thereof.
 68. Use of an inhibitor of Prohibitin for prevention of cell migration in vivo or/and in vitro.
 69. A screening method for identification of genes suitable as targets for treatment of a hyperproliferative disorder, comprising the steps (a) providing a cell or/and a transgenic non-human animal capable of so expressing, particularly overexpressing PHB, (b) downregulating PHB expression or/and inhibiting PHB activity within the cell or/and the transgenic non-human animal, (c) determining the expression or/and activity of at least one gene within the cell or/and the animal, and (d) identifying a target gene of which the expression is upregulated or downregulated by downregulation of PHB expression or/and inhibition of PHB activity.
 70. The screening method of claim 69 wherein the downregulation of PHB expression or/and the inhibition of PHB activity in step (b) is performed by an inhibitor of PHB.
 71. The screening method of claim 69, wherein in step (d) the target gene is upregulated or downregulated by a factor of at least 2, preferably at least
 4. 72. The screening method of claim 69, wherein the expression of the at least one gene in step (c) is determined by a microarray technique.
 73. A screening method for identification of a compound suitable for treatment of a hyperproliferative disorder comprising the steps (a) providing a cell or/and a transgenic non-human animal capable of expressing at least one gene selected from Table 1 or/and Table 2, (b) contacting at least one compound with the cell or/and the transgenic non-human animal, (c) determining the amount or/and the activity of the gene product of the at least one gene selected from Table 1 or/and Table 2, and (d) identifying a compound which increases the amount or/and the activity of the gene product of the at least one gene of Table 1 or decreases the amount or/and activity of the gene product of the at least one gene of Table
 2. 74. The screening method of claim 69, wherein the hyperproliferative disorder is selected from tumours, metastatic tumours, benign tumours, carcinoma, neoplastic carcinoma, gastric carcinoma, larynx carcinoma, neoplastic thyroid cancer, hepatocellular carcinoma, hyperplasia, adenocarcinoma, bladder carcinoma, EGFR overexpressing tumours, Her-2 family overexpressing tumours, Herceptin resistant tumours, B-Raf transformed tumours, and Raf-1 (C-Raf) transformed tumours.
 75. The screening method of claim 69, wherein in step (a) the cell is a mammalian cell.
 76. The screening method wherein the downregulation of PHB expression or/and the inhibition of PHB activity in step (b) is performed by an inhibitor as defined in claim
 39. 